Bacterial artificial chromosome construct encoding recombinant coronavirus

ABSTRACT

The present invention relates to methods of preparing a DNA comprising steps, wherein (a) a DNA comprising a full length copy of the genomic RNA (gRNA) or an RNA virus; or (b) a DNA comprising one or several fragments of a gRNA of an RNA virus, which fragments code for an RNA dependent RNA polymerase and at least one structural or non-structural protein; or (c) a DNA having a homology of at least 60% to the sequences of (a) or (b); is cloned into a bacterial artificial chromosome (BAC). Additionally, DNAs are provided, which comprise sequences derived from the genomic RNA (gRNA) of a coronavirus which sequences have a homology of at least 60% to the natural sequence of the virus and code for an RNA dependent RNA polymerase and at least one structural or no-structural protein, wherein a fragment of said DNA is capable of being transcribed into RNA which RNA can be assembled to a virion. Further, the use of these nucleic acids for preparation of viral RNA or virions as well as pharmaceutical preparations comprising these DNAs, viral RNAs or virions is disclosed.

This application is a national stage entry filed under 35 U.S.C. 371 ofPCT/EP00/12063, filed Nov. 30, 2000.

FIELD OF THE INVENTION

This invention relates to methods of preparing a DNA or an RNA, nucleicacids obtainable by this method and their use as vaccines and for genetherapy.

BACKGROUND OF THE INVENTION

Advances in recombinant DNA technology have led to progress in thedevelopment of gene transfer between organisms. At this time, numerousefforts are being made to produce chemical, pharmaceutical, andbiological products of economic and commercial interest through the useof gene transfer techniques.

One of the key elements in genetic manipulation of both prokaryotic andeukaryotic cells is the development of vectors and vector-host systems.In general, a vector is a nucleic acid molecule capable of replicatingor expressing in a host cell. A vector-host system can be defined as ahost cell that bears a vector and allows the genetic information itcontains to be replicated and expressed.

Vectors have been developed from viruses with both DNA and RNA genomes.Viral vectors derived from DNA viruses that replicate in the nucleus ofthe host cell have the drawback of being able to integrate into thegenome of said cell, so they are generally not very safe. In contrast,viral vectors derived from RNA viruses, which replicate in the cytoplasmof the host cell, are safer than those based on DNA viruses, since thereplication occurs through RNA outside the nucleus. These vectors arethus very unlikely to integrate into the host cell's genome.

cDNA clones have been obtained from single-chain RNA viruses withpositive-polarity [ssRNA(+)], for example, picornavirus (Racaniello &Baltimore, 1981); bromovirus (Ahlquist et al., 1984); alphavirus, agenus that includes the Sindbis virus; Semliki Forest virus (SFV) andthe Venezuelan equine encephalitis virus (VEE) (Rice et al., 1987;Liljeström and Garoff, 1991; Frolov et al., 1996; Smerdou andLiljestrom, 1999); flavivirus and pestivirus (Rice and Strauss, 1981;Lai et al., 1991; Rice et al., 1989); and viruses of the Astroviridaefamily (Geigen-muller et al., 1997). Likewise, vectors for theexpression of heterologous genes have been developed from clones of DNAcomplementary to the genome of ssRNA(+) virus, for example alphavirus,including the Sindbis virus, Semliki Forest virus (SFV), and theVenezuelan equine encephalitis (VEE) virus (Frolov et al., 1996;Liljeström, 1994; Pushko et al., 1997). However, all methods ofpreparing recombinant viruses starting from RNA viruses are stillcomplicated by the fact that most of the viruses comprise sequenceswhich are toxic for bacteria. Preparing a cDNA of the viral RNA andsubcloning of the cDNA in bacteria therefore often leads to deletion orrearrangement of the DNA sequences in the bacterial host. For thispurpose most of the commonly used subcloning and expression vectorscannot be used for preparation of large DNA sections derived fromrecombinant RNA viruses. However, obtaining vectors, which can carrylong foreign DNA sequences is required for a number of aspects in thedevelopment of pharmaceuticals, specifically vaccines.

The coronaviruses are ssRNA(+) viruses that present the largest knowngenome for an RNA virus, with a length comprised between about 25 and 31kilobases (kb) (Siddell, 1995; Lai & Cavanagh, 1997; Enjuanes et al.,1998). During infection by coronavirus, the genomic RNA (gRNA)replicates and a set of subgenomic RNAs (sgRNA) of positive and negativepolarity is synthesized (Sethna et al., 1989; Sawicki and Sawicki, 1990;van der Most & Spaan, 1995). The synthesis of the sgRNAs is anRNA-dependent process that occurs in the cytoplasm of the infected cell,although its precise mechanism is still not exactly known.

The construction of cDNAs that code defective interfering (DI) genomes(deletion mutants that require the presence of a complementing virus fortheir replication and transcription) of some coronaviruses, such as themurine hepatitis virus (MHV), infectious bronchitis virus (IBV), bovinecoronavirus (BCV) (Chang et al., 1994), and porcine gastroenteritisvirus (TGEV) (Spanish Patent Application P9600620; Méndez et al., 1996;Izeta et al., 1999; Sánchez et al., 1999) has been described. However,the construction of a cDNA clone that codes a complete genome of acoronavirus has not been possible due to the large size of and the toxicsequences within the coronavirus genome.

In summary, although a large number of viral vectors have been developedto replicate and express heterologous nucleic acids in host cells, themajority of the known vectors for expression of heterologous genes arenot well suited for subcloning of RNA viruses. Further, the viralvectors so obtained present drawbacks due to lack of species specificityand target organ or tissue limitation and to their limited capacity forcloning, which restricts the possibilities of use in both basic andapplied research.

Hence there is a need for methods to develop new vectors for expressionof heterologous genes that can overcome the aforesaid problems. Inparticular, it would be advantagous to have large vectors for expressionof heterologous genes with a high level of safety and cloning capacity,which can be designed so that their species specificity and tropism canbe controlled.

SUMMARY OF THE INVENTION

According to the present invention the above problems are solved by amethod of preparing a DNA comprising steps, wherein

-   (a) a DNA comprising a full length copy of the genomic RNA (gRNA) of    an RNA virus; or-   (b) a DNA comprising one or several fragments of a gRNA of an RNA    virus, which fragments code for an RNA dependent RNA polymerase and    at least one structural or non-structural protein; or-   (c) a DNA having a homology of at least 60% to the sequences of (a)    or (b); is cloned into a bacterial artificial chromosome (BAC).

Surprisingly, the present inventors found that the problems encounteredby the prior art methods to subclone and express large DNA sequencesderived from viral gRNA can be overcome by using BACs as a cloningvector. The use of BACs has the particular advantage that these vectorsare present in bacteria in a number of one or two copies per cell, whichconsiderably limits the toxicity and reduces the possibilities ofinterplasmid recombinantion.

The invention further provides methods of preparing a viral RNA or avirion comprising steps, wherein a DNA is prepared according to one ofthe above methods, the DNA is expressed and the viral RNA or the virionis isolated. Further, methods of preparing pharmaceuticals, specificallyvaccines comprising the steps of the above methods to prepare a DNA aredisclosed.

According to another aspect of the present invention provides a DNAcomprising sequences derived from the genomic RNA (gRNA) of acoronavirus which sequences have a homology of at least 60% to thenatural sequence of the coronavirus and code for an RNA dependent RNApolymerase and at least one structural or non-structural protein,wherein a fragment of said DNA is capable of being transcribed into RNAand which RNA can be assembled to a virion. The present invention alsoencompasses methods of preparing respective DNAs.

The present invention further provides vectors, more specificallybacterial artificial chromosomes (BACs) comprising respective nucleicacids. According to a further embodiment the present invention isdirected to host cells and infectious, attenuated or inactivated virusescomprising the DNAs or RNAs of the present invention.

The invention also provides pharmaceutical preparations, such as mono-or multivalent vaccines comprising nucleic acids, vectors, host cells orvirions of the present invention.

Finally, the present invention provides methods for producing a virionor a viral RNA comprising steps, wherein a DNA according to the presentinvention is transcribed and the virions or viral RNAs are recovered, aswell as viral RNAs obtainable by this method.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the construction of a cDNA clone that codes an infectiveRNA of TGEV.

FIG. 1A shows the genetic structure of the TGEV, with the names of thegenes indicated by letters and numbers (1a, 1b, S, 3a, 3b, E, M, N, and7).

FIG. 1B shows the cDNA-cloning strategy, which consisted in completingthe DI-C genome. Deletions Δ1, Δ2, and Δ3 that have been completed toreestablish the full length of the cDNA are indicated. The numberslocated beneath the structure of the DI-C genome indicate thenucleotides that flank each deletion in said DI-C genome.

FIG. 1C shows the four cDNA fragments constructed to complete deletionΔ1 and the position of the principal restriction sites used duringjoining. The insertion of fragment Δ1 produced an increase in thetoxicity of the cDNA.

FIG. 2 shows the structure of the pBeloBAC plasmid (Wang et al., 1997)used in cloning the infective cDNA of TGEV. The pBeloBAC plasmid wasprovided by H. Shizuya and M. Simon (California Institute of Technology)and includes 7,507 base pairs (bp) that contain the replication originof the F factor of E. coli (oriS), the genes necessary to keep onesingle copy of the plasmid per cell (parA, parB, parC, and repE), andthe chloramphenicol-resistance gene (CM^(r)). The positions of the T7and SP6 promoters and of the unique restriction sites are indicated.CosN: site cosN of lambda to facilitate the construction of the pBACplasmid; lac Z: β-galactosidase gene. Sequence loxP used during thegeneration of the plasmid is also indicated.

FIG. 3 shows the structure of the basic plasmids used in theconstruction of TGEV cDNA. The pBAC-TcDNA^(ΔClaI) plasmid contains allthe information of the TGEV RNA except for one ClaI-ClaI fragment of5,198 bp. The cDNA was cloned under the immediately early (IE) promoterof expression of cytomegalovirus (CMV) and is flanked at the 3′-end by apoly(A) tail with 24 residues of A, the ribozyme of the hepatitis deltavirus (HDV), and the termination and polyadenylation sequences of bovinegrowth hormone (BGH). The pBAC-B+C+D5′ plasmid contains the ClaI-ClaIfragment required to complete the pBAC-TcDNA^(ΔClaI) until the cDNA isfull length. The pBAC-TcDNA^(FL) plasmid contains the full-length cDNAof TGEV. SAP: shrimp alkaline phosphatase.

FIG. 4 shows the differences in the nucleotide sequence of the S gene ofthe clones of TGEV PUR46-MAD (MAD) (SEQ ID NOS: 8 & 9) and C11 (SEQ IDNOS: 6 & 7). The numbers indicate the positions of the substitutednucleotides, considering as nucleotide one of each gene the A of theinitiating codon. The letters within the bars indicate the correspondingnucleotide in the position indicated. The letters located beneath thebars indicate the amino acid (aa) substitutions coded by the nucleotidesthat are around the indicated position. Δ6 nt indicates a 6-nucleotidedeletion. The arrow indicates the position of the termination codon ofthe S gene.

FIG. 5 shows the strategy followed to rescue the infective TGEV from thefull-length TGEV cDNA. The pBAC-TcDNA^(FL) plasmid was transfected to STcells (pig testicle cells), and 48 h after transfection, the supernatantwas used to infect new ST cells. The virus was passed at the timesindicated. At each passage, aliquots of supernatant and of cellularmonolayer were collected for virus titration and isolation of RNA forRT-PCR analysis, respectively. vgRNA: full-length viral RNA.

FIG. 6 shows the cytopathic effect (CPE) produced by the TGEV cDNA inthe transfected ST cells. The absence of CPE in non-transfected(control) ST cells (FIG. 6A) and the CPE observed 14 and 20 h aftertransfection with pBAC-TcDNA^(FL) in ST cells are shown (FIGS. 6B and6C, respectively).

FIG. 7 shows the evolution of the viral titer with the passage. A graphrepresenting the viral titer in the supernatant of two series ofcellular monolayers (1 and 2) at different passages after transfectionwith pBAC-TcDNA^(FL) is shown. Mock 1 and-2 refer to nontransfected STcells. TcDNA 1 and 2 refer to ST cells transfected with pBAC-TcDNA^(FL).

FIG. 8 shows the results of the analysis of the sequence of the virusrecovered after transfecting ST cells with pBAC-TcDNA^(FL). Thestructure of the TGEV genome is indicated at the top of the figure.Likewise, the differences in the sequence of nucleotides (geneticmarkers) between the virus recovered from the pBAC-TcDNA^(FL) (TcDNA)plasmid, and TGEV clones C8 and C11 are indicated. The positions of thedifferences between the nucleotides are indicated by the numbers locatedover the bar. The cDNA sequences of the TcDNA virus and of clone C11were determined by sequencing the fragments obtained by RT-PCR(reverse-transcription and polymerase chain reaction). The sequence ofclone C8 is being sent for publication (Penzes et al., 1999) and isincluded at the end of this patent. The restriction patterns are shownwith ClaI and DraIII of the fragments obtained by RT-PCR that includenucleotides 18,997 and 20,990 of the TcDNA and C8 viruses. Therestriction patterns show the presence or absence of ClaI and DraIIIsites in the cDNA of these viruses. The result of this analysisindicated that the TcDNA virus recovered had the S-gene sequenceexpected for isolate C11. MWM: molecular weight markers.

FIG. 9 shows the results of the RT-PCR analysis of the virus recovered.The viral RNA was expressed under the control of the CMV promoterrecognized by the cellular polymerase pol II. In principle, this RNAcould undergo splicing during its transport to the cytoplasm. To studywhether this was the case, the sites of the RNA with a high probabilityof splicing were determined using a program for predicting splicingsites in sequences of human DNA (Version 2.1.5.94, Department of CellBiology, Baylor College of Medicine) (Solovyev et al., 1994). Thepotential splicing site with maximum probability of cut had the donorsite at nt 7,243 and the receiver at nt 7,570 (FIG. 9A). To studywhether this domain had undergone splicing, a RT-PCR fragment flanked bynt 7,078 and nt 7,802 (FIG. 9B) was prepared from RNA of passages 0 and2 of nontransfected cultures (control), or from ST cells transfectedwith TcDNA with the ClaI fragment in reverse orientation(TcDNA^(FL(−ΔClaI)RS)), or in the correct orientation (TcDNA^(FL)), andthe products resulting from the RT-PCR were analyzed in agarose gels.The results obtained are shown in FIGS. 9C (passage 0) and 9D (passage2).

FIG. 10 shows the results of the immunofluorescence analysis of thevirus produced in cultures of ST cells transfected with TcDNA. Stainingfor immunofluorescence was done with antibodies specific for the TGEVPUR46-MAD isolate, and for the virus recovered after transfection withthe pBAC-TcDNA^(FL) plasmid. For this, TGEV-specific monoclonalantibodies were used which bind to both isolates or only to PUR46-MAD(Sánchez et al., 1990). The result confirmed that the TcDNA virus hadthe expected antigenicity. The specific polyclonal antiserum for TGEVbound to both viruses, but not to the uninfected cultures, and only theexpected monoclonal antibodies specific for the S (ID.B12 and 6A.C3), M(3B.B3), and N (3B.D8) proteins bound to the TcDNA virus (Sánchez etal., 1999).

FIG. 11 (SEQ ID NOS: 10-15)shows the expression of GUS under differenttranscription-regulatory sequences (TRSs) that vary flanking region 5′of the intergenic (IG) sequence. Minigenome M39 was cloned under thecontrol of the CMV promoter. ( FIG. 11A.) Inserted into this minigenomewas a multiple cloning sequence (PL1, 5′-CCTAGGATTTAA-ATCCTAAGG-3′; SEQID NO: 2) and the transcription unit formed by the selectedtranscription-regulating sequences (TRS), another multiple cloningsequence (PL2, 5′-GCGGCCGCGCCGGCGAGGCCTGTCGAC-3′; SEQ ID NO:3; or PL3,5′-GTCGAC-3′; SEQ ID NO:4), sequences with the structure of a Kozak (Kz)domain, the β-glucuronidase (GUS) gene, and another multiple cloningsite (PL4, 5′-GCTAGCCCAGGCGCGCGGTACC-3′; SEQ ID NO: 5). These sequences¹were flanked at the 3′-end by the 3′-sequence of minigenome M39, theHDV ribozyme, and the termination and polyadenylation sequences of BGH.The TRSs had a different number (0, −3, −8, and −88) of nucleotides ofthe 5′-end of the IG sequence (CUAAAC)¹, and came from the N, S, or Mgenes, as indicated. ST cells were transfected with the differentplasmids, were infected with the complementing virus (PUR46-MAD), andthe supernatants were passed 6 times. The GUS activity in the infectedcells was determined by means of the protocol described by Izeta (Izetaet al., 1999). The results obtained by relating the GUS activity to thepassage number are collected in FIG. 11B. ¹ It should be noted thatCTAAAC and CUAAC have the same meaning for the purpose of this patent.The first represents the sequence of the DNA and the second that of thecorresponding RNA.

FIG. 12 (SEQ ID NOS: 16-24) shows the expression of GUS under differentTRSs that vary in the 3′-flanking region of the IG sequence (see FIG.11A). Using this transcription unit with the 5′-flanking regioncorresponding to the -88 nt of the N gene of TGEV plus the IG sequence(CUAAAC), the 3′-flanking sequences were modified. These sequencescorresponded to those of the different TGEV genes (S, 3a, 3b, E, M, N,and 7), as is indicated in FIG. 12A. In two cases, 3′-sequences werereplaced by others that contained a restriction site (SalI) and anoptimized Kozak sequence (Kz), or by a sequence identical to the onethat follows the first IG sequence located following the leader of theviral genome. The activity of GUS in the infected cells was determinedby means of the protocol described above (Izeta et al., 1999). cL12indicates a sequence of 12 nucleotides identical to that of 3′-end ofthe “leader” sequence of the TGEV genome (see the virus sequenceindicated at the end). The results obtained by relating the expressionof GUS to the passage number are collected in FIG. 12B.

FIG. 13 shows the effect of the site of insertion of the module ofexpression in the minigenome over the levels of GUS expression. The GUStranscription unit, containing −88 nt of the N gene flanking the 5′-endof the IG sequence (CUAAAC), and the Kz sequences flanking the 3′-end(see FIG. 12A), was inserted into four single restriction sites inminigenome M39 (FIG. 13A) to determine if all these sites were equallypermissive for the expression of the heterologous gene. ST cells weretransfected with these plasmids and infected with the complementingvirus (PUR46-MAD). The GUS activity in the infected cells was determinedat passage 0 (P0) following the protocol described above (Izeta et al.,1999). The results obtained are collected in FIG. 13B.

FIG. 14 (SEQ ID No: 1) shows the consent sequence of the PUR46-MADisolate of TGEV.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention methods of preparing a DNA areprovided, which comprise steps, wherein

-   -   (a) a DNA comprising a full length copy of the genomic RNA        (gRNA) of an RNA virus; or    -   (b) a DNA comprising one or several fragments of a gRNA of an        RNA virus, which fragments code for an RNA dependent RNA        polymerase and at least one structural or non-structural        protein; or    -   (c) a DNA having a homology of at least 60% to the sequences        of (a) or (b);        is cloned into a bacterial artificial chromosome (BAC).

According to the present application a “bacterial artificial chromosome”is a DNA sequence which comprises the sequence of the F factor. Plasmidscontaining this sequences, so-called F plasmids, are capable of stablymaintaining heterologous sequences longer than 300 Kb in low copy number(one or two copies per cell). Respective BACs are known in the art(Shizuya et al., 1992).

According to the present invention the DNA cloned into the BAC has ahomology of at least 60%, preferably 75% and more preferably 85 or 95%,to a natural sequence of an RNA virus. Sequence homology is preferablydetermined using the Clustal computer program available from theEuropean Bioinformatics Institute (EBI).

According to the methods of the present invention the DNA cloned intothe BAC may further comprise sequences coding for several or all exceptone of the structural or non-structural proteins of the virus.

In a preferred embodiment of the present invention the DNA cloned intothe BAC further comprises sequences encoding one or several heterologousgene. According to the present application a gene is characterized as a“heterologous gene” if it is not derived from the virus which was usedas a source for the genes encoding the RNA dependent RNA polymerase andthe structural or non-structural protein. A “heterologous gene” thusalso refers to genes derived from one type of virus and expressed in avector comprising sequences derived from another type of virus. Anyheterologous gene of interest can be inserted into the nucleic acids ofthe present invention. The insertion of genes encoding one or severalpeptides or proteins which are recognised as an antigen from aninfectious agent by the immune system of a mammal is especiallypreferred. Alternatively, the method of the present invention may beperformed using heterologous genes encoding at least one moleculeinterfering with the replication of an infectious agent or an antibodyproviding protection against an infectious agent. The heterologoussequences may contain sequences encoding an immune modulator, acytokine, an immonenhancer and/or an anti-inflammatory compound.

The method of the present invention may be performed using a DNA forcloning into a BAC that has any size. However, specific advantages overthe known methods to prepare subcloned DNA from viral are obtained, iflarge sequences are used. The DNA cloned into the BAC may thus comprisea length of at least 5 Kb, wherein DNA with a size of at least 15, 25 or30 Kb is specifically preferred.

According to specifically preferred embodiments of the present inventionmethods are provided, wherein the BAC comprises a sequence controllingthe transcription of the DNA cloned into the BAC. This will allowtranscription of the viral RNA and thus enable expression of the virus.Any sequence controlling transcription known in the art may be used forthis purpose, including sequences driving the expression of genesderived from DNA or RNA genomes. The use of the immediately early (IE)promoter of cytomegalovirus (CMV) is preferred.

The DNA cloned into the BAC may also be flanked at the 3′-end by apoly(A)tail. The nucleic acid may comprise termination and/orpolyadenylation sequences of bovine growth hormone (BGH). Additionallyor alternatively, the nucleic acids may comprise sequences encoding aribozyme, for example the ribozyme of the hepatitis δ virus (HDV).

Additional advantages may be achieved if at least one of the genes ofthe virus has been modified by substituting, deleting or addingnucleotides. For example the gene controlling tropism of the virus maybe modified to obtain viruses with altered tropism. Alternativly, thegene controlling tropism of the virus has been substituted with therespective gene of another virus. The modification is preferablyperformed in the S, M and/or N genes of the virus.

In a preferred embodiment of the present invention a method is provided,wherein the DNA cloned into the BAC is capable of being transcribed intoRNA which RNA can be assembled to an virion. The virion may be aninfectious, attenuated, replication defective or inactivated virus.

Any RNA virus may be used in the methods of the invention. The virus canfor example be a picornavirus, flavivirus, togavirus, coronavirus,toroviruses, arterivurses, calcivirus, rhabdovirus, paramixovirus,filovirus, bornavirus, orthomyxovirus, bunyavirus, arenavirus orreovirus. The use of viruses naturally having a plus strand genome ispreferred.

Additionally, the present invention provides methods of preparing aviral RNA or a virion comprising steps, wherein a DNA is preparedaccording to one of above methods, the DNA is expressed in a suitablehost cell and the viral RNA or the virion is isolated from that hostcell. Any of methods for isolating viruses from the cell culture knownin the art may be used. Alternatively, methods of preparing a viral RNAor a virion are disclosed, wherein the DNA of the present invention istranscribed or translated using chemicals, biological reagents and/orcell extracts and the viral RNA or the virion is subsequently isolated.For certain embodiments, the virus may subsequently be inactivated orkilled.

The invention also provides methods for preparing a pharmaceuticalcomposition comprising steps, wherein a DNA, a viral RNA or a virion isprepared according to one of the above methods and is subsequently mixedwith a pharmaceutically acceptable adjuvans and/or carrier. A largenumber of adjuvans and carriers and diluents are known in the prior artand may be used in accordance with the present invention. Thepharmaceutical is preferably a vaccine for protecting humans or animalsagainst an infectious disease. The pharmaceutical can advantageouslyalso be used for gene therapy.

The present invention further provides for the first time a DNAcomprising sequences derived from the genomic RNA (gRNA) of acoronavirus which sequences have a homology of at least 60% to thenatural sequence of the coronavirus and code for an RNA dependent RNApolymerase and at least one structural or non-structural protein,wherein a fragment of said DNA is capable of being transcribed into RNAwhich can be assembled to a virion.

According to the present invention the term “sequence derived from acoronavirus” is used to refer to a nucleic acid sequence which has ahomology of at least 60%, preferably 75% and more preferably 85 or 95%,to a natural sequence of a coronavirus. Sequence homology can bedetermined using the Clustal computer program available from theEuropean Bioinformatics Institute (EBI).

The term “coronavirus” is used according to the present invention torefer to a group of viruses having a single molecule of linear, positivesense, ssRNA of 25 to 33 Kb. These viruses usually contain 7 to 10structural genes, i.e. genes encoding proteins that determine the viralstructure. These genes are typically arranged in the viral genome in theorder of 5′replicase-(hemagglutinin-esterase)-spike-envelope-membrane-nucleoprotein-3′.Additionally the viral genome may comprise a number of non-structuralgenes which encode a nested set of mRNAs with a common 3′ end and arelargely non-essential.

The term “capable of being transcribed into RNA which can be assembledinto a virion” is used to characterize a DNA sequence, which—onceintroduced into a suitable host cell—will be transcribed into RNA andgenerate virions. The virions are preferably infectious viruses, but mayalso be inactivated, attenuated or replication defective virusescomprising said RNA. Preferably all the information necessary forexpression of the virion is encoded by the DNA sequence of the presentinvention.

The nucleic acids of the present invention may further comprise asequence encoding one or several heterologous genes of interest.According to the present invention a gene is characterized as a“heterologous gene” if it is not derived from the coronavirus which wasused as a source for the genes encoding the RNA dependent RNA polymeraseand the structural or non-structural protein. A “heterologous gene” thusalso refers to genes derived from one type of coronavirus and expressedin a vector comprising sequences derived from another type ofcoronavirus. Any heterologous gene of interest can be inserted into thenucleic acids of the present invention. The insertion of genes encodingpeptides or proteins which are recognised as an antigen from aninfectious agent by the immune system of a mammal is especiallypreferred. The heterologous gene may thus encode at least one antigensuitable for inducing an immune response against an infectious agent, atleast one molecule interfering with the replication of an infectiousagent or an antibody providing protection against an infectious agent.Alternatively or additionally, the heterologous gene may encode animmune modulator, a cytokine, an immonenhancer or an anti-inflammatorycompound.

The fragment of the DNA according to the present invention which istranscribed into RNA preferably has a size of at least 25 Kb. Fragmentswith a size of at least 30 Kb are especially preferred.

According to a preferred embodiment of the present invention the DNAfurther comprises sequences derived from a coronavirus coding forseveral or all except one of the structural or nonstructural proteins ofa coronavirus. Alternatively, the DNA of the present invention furthercomprises sequences coding for all of the structural or non-structuralproteins of a coronavirus.

According to a further embodiment, the nucleic acids of the presentinvention comprise a sequence controlling the transcription of asequence derived from a coronavirus gRNA. Any sequence controllingtranscription known in the art may be used for this purpose, includingsequences driving the expression of genes derived from DNA or RNAgenomes. The use of the immediately early (IE) promoter ofcytomegalovirus (CMV) is preferred.

The nucleic acid according to the present invention may also be flankedat the 3′-end by a poly(A)tail. The nucleic acid may comprisetermination and/or polyadenylation sequences of bovine growth hormone(BGH). Additionally or alternatively, the nucleic acids may comprisesequences encoding a ribozyme, for example the ribozyme of the hepatitisδ virus (HDV).

The nucleic acids of the present invention may comprise sequencesderived from any coronavirus, for example derived from an isolate of theporcine transmissible gastroenteritis virus (TGEV), murine hepatititsvirus (MHV), infectious bronchitis virus (IBV), bovine coronavirus(BoCV), canine coronavirus (CCoV), feline coronavirus (FcoV) or humancoronavirus. According to a preferred embodiment the nucleic acid isderived from a transmissable gastroenteritis virus.

According to a further embodiment of the present invention, the DNAs ofthe present invention are part of a plasmid, preferably part of abacterial artificial chromosome (BAC).

The present invention further provides host cells comprising respectivenucleic acids or vectors. The host cells may be eucaryotes orprocaryotes. Alternatively, the present invention provides virionscomprising a nucleic acid according the present invention. Respectivevirions may for example be isolated from cell cultures transfected orinfected with the nucleic acids of the present invention.

According to a further embodiment, the present invention providesmethods for producing a virion or a viral RNA comprising steps, whereina DNA of the present invention is introduced into a host cell, hostcells containing the DNA are cultivated under conditions allowing theexpression thereof and the virion or viral RNA is recovered.Additionally, methods for producing a virion or a viral RNA areprovided, wherein a DNA of the present invention is mixed in vitro withchemicals, biological reagents and/or cell extracts under conditionsallowing the expression of the DNA and the virion or viral RNA isrecovered. The present invention also encompasses the virions and viralRNAs obtainable by either of the above methods. RNAs and virionscarrying a heterologous gene are preferred. The viruses so obtained mayhave the form of an infectious, attenuated, replication defective orinactivated virus.

The virus may comprise modified genes, for example a modified S, N or Mgene. In a specific embodiment of the present invention the modificationof the S, N or M gene gives raise to an attenuated virus or a virus withaltered tropism.

According to a further embodiment the invention provides apharmaceutical preparation comprising nucleic acids, host cells orvirions according to the present invention. According to a preferredembodiment the pharmaceutical preparation is a vaccine capable ofprotecting an animal against diseases caused by an infectious agent. Thevaccine may for example comprise sequences of at least one antigensuitable for inducing an immune response against the infectious agent oran antibody providing protection against said infectious agent. Thevaccine may comprise a DNA expressing at least one molecule interferingwith the replication of the infectious agent. Alternatively the vaccinemay comprise a vector expressing at least one antigen capable ofinducing a systemic immune response and/or an immune response in mucousmembranes against different infectious agents that propagate inrespiratory, intestinal mucous membranes or in other tissues. Thevaccine may also be a multivalent vaccine capable of protecting ananimal against the infection caused by more than one infectious agent,that comprises more than one nucleic acid of the present invention eachof which expresses an antigen capable of inducing an immune responseagainst each of said infectious agents, or antibodies that provideprotection against each one of said infectious agents or other moleculesthat interfere with the replication of any infectious agent.

The vaccines of the present invention may further comprise any of thepharmaceutically acceptable carriers or diluents known in the state ofthe art.

The present invention further provides methods for preparing a DNA ofthe present invention comprising steps, wherein an interfering defectivegenome derived from a coronavirus is cloned under the expression of apromotor into a BAC vector and the deletions within the defective genomeare re-inserted. The method may further comprise steps, wherein toxicsequences within the viral genome are identified before re-insertioninto the remaining genomic DNA. Preferably, the toxic sequences withinthe viral genome are the last sequences to be re-inserted beforecompleting the genome. According to the present invention this method issuitable to yield infectious clones of coronaviruses which are stable inbacteria for at least 80 generations and thus provides a very efficientcloning vector.

The present invention provides the development of infective clones ofcDNA derived from coronavirus (Almazan et al., 2000), as well as vectorsconstructed from said infective clones that also include heterologousnucleic acid sequences inserted into said clones. The infective clonesand vectors provided by this invention have numerous applications inboth basic and applied research, as well as a high cloning capacity, andcan be designed in such a way that their species specificity and tropismcan be easily controlled.

This patent describes the development of a method that makes it possibleto obtain, for the first time in the history of coronavirus, afull-length infective cDNA clone that codes the genome of a coronavirus(Almazan et al., 2000).

A new vector or system of expression of heterologous nucleic acids basedon a coronavirus generated from an infective cDNA clone that codes thegenomic RNA (gRNA) of a coronavirus has been developed. In oneparticular realization of this invention, the coronavirus is the porcinetransmissible gastroenteritis virus (TGEV).

The new system of expression can be used in basic or applied research,for example, to obtain products of interest (proteins, enzymes,antibodies, etc.), as a vaccinal vector, or in gene therapy in bothhumans and animals. The infective coronavirus obtained from theinfective cDNA clone can be manipulated by conventional geneticengineering techniques so that new genes can be introduced into thegenome of the coronavirus, and so that these genes can be expressed in atissue- and species-specific manner to induce an immune response or forgene therapy. In addition, the expression has been optimized by theselection of new transcription-regulating sequences (TRS) that make itpossible to increase the levels of expression more than a hundredfold.

The vectors derived from coronavirus, particularly TGEV, present severaladvantages for the induction of immunity in mucous membranes withrespect to other systems of expression that do not replicate in them:(i) TGEV infects intestinal and respiratory mucous membranes (Enjuanesand Van der Zeijst, 1995), that is, the best sites for induction ofsecretory immunity; (ii) its tropism can be controlled by modifying theS (spike) gene (Ballesteros et al., 1997); (iii) there are nonpathogenicstrains for the development of systems of expression that depend oncomplementing virus (Sánchez et al., 1992); and (iv) coronaviruses arecytoplasmic RNA viruses that replicate without passing through anintermediate DNA stage (Lai and Cavanagh, 1997), making its integrationinto the cellular chromosome practically impossible.

The procedure that has made it possible to recover an infectivecoronavirus from a cDNA that codes the gRNA of a coronavirus includesthe following strategies:

-   (i) expression of the RNA of the coronavirus under the control of an    appropriate promoter;-   (ii) cloning of the genome of the coronavirus in bacterial    artificial chromosomes (BACs);-   (iii) identification of the sequences of cDNA of the coronavirus    that are directly or indirectly toxic to bacteria;-   (iv) identification of the precise order of joining of the    components of the cDNA that codes an infective RNA of coronavirus    (promoters, transcription-termination sequences, polyadenylation    sequences, ribozymes, etc.); and-   (V) identification of a group of technologies and processes    (conditions for the growth of BACs, modifications to the    purification process of BAC DNA, transformation techniques, etc.)    that, in combination, allow the efficient rescue of an infective    coronavirus of a cDNA.

The promoter plays an important role in increasing the expression ofviral RNA in the nucleus, where it is synthesized, to be transported tothe cytoplasm later on.

The use of BACs constitutes one of the key points of the procedure ofthe invention. As is known, cloning of eukaryotic sequences in bacterialplasmids is often impossible due to the toxicity of the exogenoussequences for bacteria. In these cases, the bacteria usually eliminatetoxicity by modifying the introduced sequences. Nevertheless, in thestrategy followed in this case, to avoid the possible toxicity of theseviral sequences, the necessary clonings were carried out to obtain acomplete cDNA of the coronavirus in BACs. These plasmids appear in onlyone copy or a maximum of two per cell, considerably limiting theirtoxicity and reducing the possibilities of interplasmid recombination.

Through the identification of the bacteriotoxic cDNA sequences of thecoronavirus, the construction of the cDNA that codes the complete genomeof a coronavirus can be completed, with the exception of the toxicsequences, which are added in the last step of construction of thecomplete genome, that is, just before transfection in eukaryotic cells,avoiding their modification by the bacteria.

One object of the present invention therefore consists in an infectivedouble-chain cDNA clone that codes the gRNA of a coronavirus, as well asthe procedure for obtaining it.

An additional object of this invention consists in a set of recombinantviral vectors that comprises said infective clone and sequences ofheterologous nucleic acids inserted into said infective clone.

An additional object of this invention consists in a method forproducing a recombinant coronavirus that comprises the introduction ofsaid infective clone into a host cell, the culture of the transformedcell in conditions that allow the replication of the infective clone andproduction of the recombinant coronavirus, and recovering therecombinant coronavirus from the culture.

Another object of this invention consists in a method for producing amodified recombinant coronavirus that comprises introducing therecombinant viral vector into a host cell, cultivating it in conditionsthat allow the viral vector to replicate and the modified recombinantcoronavirus to be produced, and recovering the modified recombinantcoronavirus from the culture. Another object of this invention consistsin a method for producing a product of interest that comprisescultivating a host cell that contains said recombinant viral vector inconditions that allow the expression of the sequence of heterologousDNA.

Cells containing the aforementioned infective clones or recombinantviral vector constitute another object of the present invention.

Another object of this invention consists in a set of vaccines thatprotect animals against infections caused by infectious agents. Thesevaccines comprise infective vectors that express at least one antigenadequate for inducing an immune response against each infective agent,or at least one antibody that provides protection against said infectiveagent, along with a pharmaceutically acceptable excipient. The vaccinescan be mono- or multivalent, depending on whether the vectors expressone or more antigens capable of inducing an immune response to one ormore infectious agents, or, alternatively, one or more antibodies thatprovide protection against one or more infectious agents.

Another object provided by this invention comprises a method of animalimmunization that consists in the administration of said vaccine.

The invention provides a cDNA clone that codes the infective RNA of acoronavirus, henceforth the infective clone of the invention, whichcomprises: (1) a copy of the complementary DNA (cDNA) to the infectivegenomic RNA (gRNA) of a coronavirus or the viral RNA itself; and (2) anexpression module for an additional gene, which includes optimizedtranscription-promoting sequences.

In one particular realization of this invention, the coronavirus is aTGEV isolate, in particular, the PUR46-MAD isolate (Sánchez et al.,1990), modified by the replacement of the S gene of this virus by the Sgene of the clone C11 TGEV isolate or the S-gene of a canine or humancoronavirus.

The transcription-promoting sequence, or promoter, is an RNA sequencelocated at the 5′-terminal end of each messenger RNA (mRNA) ofcoronavirus, to which the viral polymerase RNA binds to begin thetranscription of the messenger RNA (mRNA). In a particular and preferredembodiment the viral genome is expressed from a cDNA using the IEpromoter of CMV, due to the high level of expression obtained using thispromoter (Dubensky et al., 1996), and to previous results obtained inour laboratory that indicated that large defective genomes (9.7 kb and15 kb) derived from the TGEV coronavirus expressed RNAs that did notundergo splicing during their transport from the nucleus, where they aresynthesized, to the cytoplasm.

The infective clone of the invention also contains a transcriptiontermination sequence and a polyadenylation signal such as that comingfrom the BGH gene. These termination sequences have to be placed at the3′-end of the poly(A) tail. In one particular realization, the infectiveclone of the invention contains a poly(A) tail of 24 residues of A andthe termination and polyadenylation sequences of the BGH separated fromthe poly(A) tail by the sequence of the HDV ribozyme.

The plasmid into which the infective cDNA of the virus has been insertedis a DNA molecule that possesses a replication origin, and is thereforepotentially capable of replicating in a suitable cell. The plasmid usedis a replicon adequate for maintaining and amplifying the infectiveclone of the invention in an adequate host cell such as a bacterium, forexample, Escherichia coli. The replicon generally carries a gene ofresistance to antibiotics that allows the selection of the cells thatcarry it (for example, cat).

In Example 1, the construction of an infective clone of TGEV under thecontrol of the IE promoter of CMV is described. The 3′-end of the cDNAappears flanked by a 24 nt poly(A) sequence, the HDV ribozyme, and thetranscription termination sequence of the BGH.

The procedure for obtaining the infective clone of the inventioncomprises constructing the full-length cDNA from the gRNA of acoronavirus and joining the transcription-regulating elements.

The cDNA that codes the infective gRNA of a coronavirus was obtainedfrom a DI genome derived from a coronavirus cloned as a cDNA under thecontrol of an appropriate promoter in a BAC, for the purpose ofincreasing the cDNA's stability. Then the bacteriotoxic sequences wereidentified and, for the purpose of eliminating that toxicity, said toxicsequences were removed and inserted at the end of the construction ofthe complete genome, just before transfection in eukaryotic cells. Theviral progeny can be reconstituted by means of transfection of the BACplasmid that contains the coronavirus genome in eukaryotic cells thatsupport viral replication.

The transcription-regulating elements are joined by means ofconventional techniques (Maniatis et al., 1989).

The infective clone of the invention can be manipulated by conventionalgenetic engineering techniques to insert at least one sequence of aheterologous nucleic acid that codes a determined activity, under thecontrol of the promoter that is present in the infective clone and ofthe regulating sequences contained in the resulting expression vector.

The infective clone of the invention presents numerous applications; forexample, it can be used both in basic research, for example, to studythe mechanism of replication and transcription of coronaviruses, and inapplied research, for example, in the development of efficient systemsof expression of products of interest (proteins, enzymes, antibodies,etc.).

Appropriate cells can be transformed from the infective cDNA clone ofthe invention, and the virions obtained containing the complete genomeof the coronavirus can be recovered. Therefore, the invention moreoverprovides a method for producing a recombinant coronavirus that comprisesthe introduction of an infective cDNA of the invention into a host cell,the culture of said cell under conditions that allow the expression andreplication of the infective clone and the recovery of the virionsobtained from the recombinant coronavirus, which contain the infectivegenome of the coronavirus. The infective clone of the invention can beintroduced into the host cell in various ways, for example bytransfection of the host cell with an RNA transcribed in vitro from aninfective clone of the invention, or by infecting the host cell with theinfective cDNA clone of the invention. Said host cells that contain theinfective clone of the invention constitute an additional object of thepresent invention.

The invention also provides a set of recombinant viral vectors derivedfrom an infective clone of the invention, henceforth viral vectorsof-the invention. The viral vectors of the invention comprise aninfective cDNA clone of the invention modified to contain a heterologousnucleic acid inserted into said infective clone under conditions thatallow said heterologous nucleic acid to be expressed.

The term “nucleic acid,” as it is used in this description, includesgenes or gene fragments as well as, in general, any molecule of DNA orRNA.

In the sense used in this description, the term “heterologous” appliedto a nucleic acid refers to a nucleic acid sequence that is not normallypresent in the vector used to introduce the heterologous nucleic acidinto a host cell.

The heterologous nucleic acid that can contain the viral vector of theinvention can be a gene or fragment that codes a protein, a peptide, anepitope, or any gene product of interest (such as antibodies, enzymes,etc.). The heterologous nucleic acid can be inserted into the infectiveclone of the invention by means of conventional genetic engineeringtechniques in any appropriate region of the cDNA, for example, after ORF1b or between genes N and 7, following the initiator codon (AUG), and inreading frame with that gene; or, alternatively, in the zonescorresponding to other ORFs. In the construction of the viral vector ofthe invention, it is essential that the insertion of the heterologousnucleic acid not interfere with any of the basic viral functions.

The viral vector of the invention can express one or more activities. Inthis latter case, the viral vector will include as many sequences ofheterologous nucleic acid as activities to be expressed, preceded by oneor several promoters, or by a promoter and various ribosome recognitionsites (IRES, internal ribosome entry sites), or by various promoters andone ribosome recognition site.

Therefore, the invention provides a method for producing a product ofinterest that comprises cultivating a host cell that contains a viralvector of the invention under conditions that allow the heterologousnucleic acid to be expressed and the product of interest to berecovered. Said host cells that contain the viral vector of theinvention constitute an additional object of the present invention.

The viral vector of the invention can be designed so that its speciesspecificity and tropism can be easily controlled. Due to thesecharacteristics, a very interesting application of the viral vectors ofthe invention is their use in gene therapy as a vector of the gene ofinterest, or as a vaccinal vector to induce immune responses againstdifferent pathogens.

The invention furthermore provides vaccines, capable of protecting ananimal against the infection caused by an infectious agent, thatcomprise (i) at least one viral vector of the invention that expressesat least one antigen suitable for inducing an immune response againstsaid infectious agent, or an antibody that provides protection againstsaid infectious agent, along with, optionally, (ii) a pharmaceuticallyacceptable excipient.

In the sense used in this description, “inducing protection” should beunderstood as the immune response of the receiving organism (animal tobe immunized) induced by the viral vector of the invention, throughsuitable mechanisms such as that induced by substances that potentiatecellular response (interleukins, interferons, etc.), cellular necrosisfactors, and similar substances that protect the animal from infectionscaused by infectious agents.

Included under the term “animal” are all animals of any species,preferably mammals, including man.

The term “infectious agent” in the sense used in this descriptionincludes any viral, bacterial, fungal, parasitic, or other infectiveagent that can infect an animal and cause it a pathology.

In one particular realization, the vaccine provided by this inventioncomprises at least one viral vector of the invention that expresses atleast one antigen capable of inducing a systemic immune response and/oran immune response in mucous membranes against different infectiousagents that propagate in respiratory or intestinal mucous membranes. Thevectors of the invention are quite suitable to induce immunity in mucousmembranes as well as lactogenic immunity, which is of special interestin protecting newborns against intestinal tract infections.

In another particular realization, the vaccine provided by thisinvention comprises at least one viral vector of the invention thatexpresses at least one gene that codes for the light and heavy chains ofan antibody of any isotype (for example, IgG₁, IgA, etc.) that protectsagainst an infectious agent.

Species specificity can be controlled so that the viral vector mayexpress the S protein of the envelope of a coronavirus that infects thedesired species (man, dog, cat, pig, etc.), suitable to be recognized bythe cellular receptors of the corresponding species.

The vaccines provided by this invention can be monovalent ormultivalent, depending on whether the viral vectors of the inventionexpress one or more antigens capable of inducing an immune response toone or more infectious agents, or one or more antibodies that provideprotection against one or more infectious agents.

In a particular realization of this invention, monovalent vaccinescapable of protecting man, pigs, dogs and cats against differentinfectious human, porcine, canine, and feline agents are provided, andtropism is controlled by expressing the S glycoprotein of thecoronavirus with the desired species specificity.

The monovalent vaccines against porcine infectious agents can contain avector that expresses an antigen selected from the group consistingessentially of antigens of the following porcine pathogens:Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilusparasuis, porcine parvovirus, Leptospira, Escherichia coli,Erysipelotrix rhusiopathiae, Pasteurella multocida, Bordetellabronchiseptica, Clostridium sp., Serpulina hydiosenteriae, Mycoplasmahyopneumoniae, porcine epidemic diarrhea virus (PEDV), porcinerespiratory coronavirus, rotavirus, or against the pathogens that causeporcine respiratory and reproductive syndrome, Aujeszky's disease(pseudorabies), swine influenza, or transmissible gastroenteritis, andthe etiological agent of atrophic rhinitis and proliferative ileitis.The monovalent vaccines against canine infectious agents can contain anexpression vector that expresses an antigen selected from the groupessentially consisting of antigens of the following canine pathogens:canine herpes viruses, types 1 and 2 canine adenovirus, types 1 and 2canine parvovirus, canine reovirus, canine rotavirus, caninecoronavirus, canine parainfluenza virus, canine influenza virus,distemper virus, rabies virus, retrovirus, and canine calicivirus.

The monovalent vaccines against feline infectious agents can contain anexpression vector that expresses an antigen selected from the groupessentially consisting of antigens of the following feline pathogens:cat calicivirus, feline immunodeficiency virus, feline herpes viruses,feline panleukopenia virus, feline reovirus, feline rotavirus, felinecoronavirus, cat infectious peritonitis virus, rabies virus, felineChlamydia psittaci, and feline leukemia virus.

The vectors can express an antibody that provides protection against aninfectious agent, for example, a porcine, canine or feline infectiousagent such as those cited above. In one particular realization, thevector expresses the recombinant monoclonal antibody identified as6A.C3, which neutralizes TGEV, expressed with isotypes IgG₁ or IgA, inwhich the constant part of the immunoglobulin is of porcine origin, orneutralizing antibodies for human and porcine rotaviruses.

As the excipient, a diluent such as physiological saline or othersimilar saline solutions can be used. Likewise, these vaccines can alsocontain an adjuvant from those usually used in the formulation of bothaqueous vaccines, such as aluminum hydroxide, QuilA, suspensions ofalumina gels and the like, and oily vaccines based on mineral oils,glycerides, fatty acid derivatives, and their mixtures.

The vaccines of the present invention can also containcell-response-potentiating (CRP) substances, that is, substances thatpotentiate subpopulations of helper T-cells (Th₁ and Th₂) such asinterleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-6, IL-12, gamma-IFN(gamma-interferon), cellular necrosis factor, and similar substancesthat could theoretically provoke cellular immunity in vaccinatedanimals. These CRP substances could be used in vaccine formulations withaqueous or oily adjuvants. Another type of adjuvants that modulate andimmunostimulate cellular response can also be used, such as MDP (muramyldipeptide), ISCOM (Immunostimulant Complex), or liposomes.

The invention provides multivalent vaccines capable of preventing andprotecting animals from infections caused by different infectiousagents. These multivalent vaccines can be prepared from viral vectors ofthe invention into which the different sequences that code thecorresponding antigens have been inserted in the same recombinantvector, or by constructing independent recombinant vectors that wouldlater be mixed for joint inoculation. Therefore, these multivalentvaccines comprise a viral vector that contains more than one sequence ofheterologous nucleic acids that code for more than one antigen or,alternatively, different viral vectors, each of which expresses at leastone different antigen.

Analogously, multivalent vaccines that comprise multivalent vectors canbe prepared using sequences that code antibodies that protect againstinfectious agents, instead of sequences that code the antigens.

In one particular realization of this invention, vaccines capable ofimmunizing humans, pigs, dogs, and cats against different porcine,canine and feline infectious agents, respectively, are provided. Forthis, the viral vectors contained in the vaccine must express differentantigens of the human, porcine, canine or feline pathogens mentionedabove or others.

The vaccines of this invention can be presented in liquid or lyophilizedform and can be prepared by suspending the recombinant systems in theexcipient. If said systems were in lyophilized form, the excipientitself could be the reconstituting substance.

Alternatively, the vaccines provided by this invention can be used incombination with other conventional vaccines, either forming part ofthem or as a diluent or lyophilized fraction to be diluted with otherconventional or recombinant vaccines.

The vaccines provided by this invention can be administered to theanimal orally, nasally, subcutaneously, intradermally,intraperitoneally, intramuscularly, or by aerosol.

The invention also provides a method for the immunization of animals, inparticular pigs, dogs and cats, against one or various infectious agentssimultaneously, that comprises the oral, nasal, subcutaneous,intradermal, intraperitoneal, intramuscular, or aerosol administration(or combinations thereof) of a vaccine that contains an immunologicallyefficacious quantity of a recombinant system provided by this invention.

In addition, the invention also provides a method for protecting newbornanimals against infectious agents that infect said animals, consistingin the oral, nasal, subcutaneous, intradermal, intraperitoneal,intramuscular, or aerosol administration (or combinations thereof) of avaccine of those provided by this invention to mothers before or duringthe gestation period, or to their offspring.

The invention is illustrated by the following examples, which describein detail the obtainment of infective clones and the construction of theviral vectors of the invention. These examples should not be consideredas limiting the scope of the invention, but as illustrating it. In saidexample, the transformation and growth of bacteria, DNA purification,sequence analysis, and the-assay to evaluate the stability of theplasmids were carried out according to the methodology described below.

Transformation of Bacteria

All of the plasmids were electroporated in the E. coli DH10B strain(Gibco BRL), introducing slight modifications to previously describedprotocols (Shizuya et al., 1992). For each transformation, 2 μL of theligation and 50 μL of competent bacteria were mixed in 0.2-cm dishes(BioRad) and electroporated at 200 Ω, 2.5 kV, and 25 μF. Then, 1 mL ofSOC medium (Maniatis et al., 1989) was added at each transformation, thecells were incubated a 37° C. for 45 min, and finally, the recombinantcolonies were detected on plates of LB SOC media (Maniatis et al., 1989)with 12.5 μg/mL of chloramphenicol.

Growth Conditions of the Bacteria

The bacteria containing the original plasmids, in which the incompletegenome of TGEV was cloned (FIG. 3), were grown at 37° C., showing normalgrowth kinetics. On the other hand, the BAC that contained the completecDNA was grown at 30° C. for the purpose of minimizing instability asmuch as possible. Even so, the size of the colonies was reduced andincubation periods of up to 24 h were necessary to achieve normal colonysizes.

Purification of DNA

The protocol described by Woo (Woo et al., 1994) was followed, withslight modifications. From a single colony, 4 L of LB were inoculatedwith chloramphenicol (12.5 μg/ml). After an incubation period of 18 h at30° C., the bacteria were collected by centrifugation at 6,000 G, andthe plasmid was purified using the Qiagen Plasmid Maxipreparations kitaccording to the manufacturer's recommendations. By means of thisprocedure, it was observed that the plasmid DNA obtained wascontaminated with bacterial DNA. To eliminate the contaminatingbacterial DNA, the plasmidic DNA was purified by means of centrifugationat 55,000 rpm for 16 h on a CsCl gradient. The yield obtained wasbetween 15 and 30 μg/L, depending on the size of the plasmid.

Sequence Analysis

The DNA was sequenced in an automatic sequencer (373 DNA Sequencer,Applied Biosystems) using dideoxynucleotides marked with fluorochromesand a temperature-resistant polymerase (Perkin Elmer). The reagents wereobtained by way of a kit (ABI PRISM Dye Terminator Cycle SequencingReady Reaction Kit) from the Applied Biosystems company. Thethermocycler used to perform the sequencing reactions was a “GeneAmpPCRSystem 9600” (Perkin Elmer).

The joining of the sequences and their comparison with the consensussequence of the TGEV were carried out using the SeqMan II and Align(DNASTAR) programs, respectively. No differences in relation to theconsent sequence were detected.

Stability of the Plasmids

From the original glycerolates, the bacteria that contained recombinantpBeloBAC11 plasmids were grown in 20 mL of LB with chloramphenicol (12.5μg/mL) for 16 h at 30° C. and 37° C. This material was consideredpassage 0. The bacteria were diluted 10⁶ times and grown at 30° C. and37° C. for 16 h. Serial passages were realized during eight consecutivedays (each passage represents approximately 20 generations). The plasmidDNA was purified by Miniprep at passages 0 and 8 (160 generations) andanalyzed with restriction endonucleases. The two plasmids that containedpart of the genome of TGEV were highly stable, whereas the plasmid thatcontained the complete genome of TGEV showed a certain instability after40 generations (at this point approximately 80% of the DNA presented thecorrect restriction pattern).

EXAMPLE 1

Construction of a Recombinant Vector based on a Clone of Infective cDNADerived from TGEV

1.1 Generation of an Infective cDNA of TGEV

For the purpose of obtaining a cDNA that coded for the complete TGEVgenome, we originally started with a cDNA that coded for the defectiveDI-C genome (Méndez et al., 1996). This cDNA, with an approximate lengthof one third of the TGEV genome, was cloned in the low-copy pACNR1180plasmid (Ruggli et al., 1996) and its sequence was determined. The cDNAthat coded the defective genome was efficiently rescued (replicated andpackaged) with the help of a complementing virus (Méndez et al., 1996;Izeta et al., 1999).

The DI-C genome presents three deletions (Δ1, Δ2, and Δ3) ofapproximately 10, 1 and 8 kilobases (kb), at ORFs 1a, 1b, and betweengenes S and 7, respectively (see FIG. 1).

The strategy followed to complete the sequence of a cDNA that would codefor an infective TGEV genome was to incorporate, step by step, thesequences deleted in the DI-C genome, analyzing the bacteriotoxicity ofthe new generated constructions. This aspect is very important, since itis widely documented in the scientific literature that recombinantplasmids presenting cDNAs of RNA virus generally grew poorly and wereunstable (Boyer and Haenni, 1994; Rice et al., 1989; Mandl et al.,1997).

The first deletion to be completed was deletion Δ2, of 1 kb, of ORF 1b,yielding a stable recombinant plasmid. The sequence that lacked ORF 1awas introduced by cloning cDNA fragments A, B, C, and D (FIG. 1)(Almazan et al., 2000) in such a way that all the information requiredfor the gene of the replicase would be complete. The recombinant plasmidobtained was unstable in the bacteria, generating new plasmids that hadincorporated additions and deletions in fragment B (Almazan et al.,2000). Interestingly, the elimination of a 5,198 bp ClaI-ClaIrestriction fragment that encompassed the region of the genome comprisedbetween nucleotides 4,417 and 9,615 (Penzes et al., 1999) yielded arelatively stable plasmid in the E. coli DH10B strain. Later, thesequence of deletion Δ3 was added by cloning all the genetic informationfor the structural and nonstructural proteins of the 3′-end of the TGEVgenome (FIG. 1).

For the purpose of incrementing the stability of the TGEV cDNA, it wasdecided that it would be subcloned in BAC using the pBeloBAC11 plasmid(Kim et al., 1992) (see FIG. 2). The pBeloBAC11 plasmid was a generousgift from H. Shizuya and M. Simon (California Institute of Technology).The plasmid, 7,507 bp in size, includes the replication origin of the Ffactor from parB, parC, E. coli (oriS) and the genes necessary to keep asingle copy of the plasmid per cell (parA, and repE). The plasmid alsopresents the gene of resistance to chloramphenicol (cat) as a selectionmarker. The cDNA was cloned under the control of the IE promoter of CMV,due to the high level of expression obtained using this promoter(Dubensky et al., 1996) and to previous results obtained in ourlaboratory, indicating that large (9.7 kb and 15 kb) defective genomesderived from TGEV expressed RNAs that did not undergo splicing duringtransport from the nucleus, where they are synthesized, to the cytoplasm(Izeta et al., 1999; Penzes et al., 1999; Almazan et al., 2000). Thegenerated TGEV cDNA (pBAC-TcDNA-AClaI) contained the information for thegenes of the replicase, with the exception of the deleted 5,198 bp ClaIfragment, and all the information of the structural and nonstructuralgenes. The 3′-end of the cDNA appears flanked by a 24 nt polyA sequence,the HDV ribozyme, and the transcription termination sequence of BGH(Izeta et al., 1999). On the other hand, the ClaI fragment necessary togenerate a complete genome of TGEV was cloned in BAC, generating theplasmid pBAC-B+C+D5′, which contained the region of the TGEV genomebetween 4,310 and 9,758 (see FIG. 3). Both plasmids were grown in the E.coli DH10B strain and sequenced in their entirety. The sequence obtainedwas identical to the consent sequence of the PUR46-MAD isolate of TGEVprovided at the end of this document (SEQ ID NO: 1), with the exceptionof two replacements in the positions of nucleotides 6,752 (A=>G, silent)and 18,997 (T=>C, silent), and the changes in the S gene of thePUR46-MAD that has been replaced by the D gene of isolate C11 (thesechanges are indicated in FIG. 4).

Furthermore, for the purpose of generating a cDNA that would code avirulent TGEV, the S gene of the PUR46-MAD isolate, which replicates athighs levels in the respiratory tract (>10⁶ PFU/g of tissue) and at lowlevels in the intestinal tract (<10³ PFU/mL), was completely replaced bythe S gene of TGEV clone 11, henceforth C11, which replicates withelevated titers both in the respiratory tract (<10⁶ PFU/mL) and in theintestinal tract (<10⁶ PFU/mL) (Sánchez et al., 1999). The S gene of C11presents 14 nucleotides that differ from the S gene of the PUR46-MADisolate, plus a 6 nt insertion at the 5′-end of the S gene (see FIG. 4)(Sánchez et al., 1999). Previous results in our laboratory (Sánchez etal., 1999) showed that mutants generated by directed recombination, inwhich the S gene of the PUR46-MAD isolate of the TGEV was replaced withthe S gene of the C11 intestinal isolate, acquired intestinal tropismand increased virulence, unlike the natural PUR46-MAD isolate of theTGEV that replicates very little or not at all in the intestinal tractsof infected pigs.

A cDNA was constructed from the PUR46-MAD isolate of TGEV with the Sgene of the intestinal isolate C11, by means of cloning of the 5,198 bpClaI-ClaI fragment, obtained from the pBAC-B+C+D5′ plasmid, in thepBAC-TcDNA^(−ΔClaI) plasmid, to generate the pBAC-TcDNA^(FL) plasmidthat contains the cDNA that codes for the complete TGEV genome (FIG. 3).

The stability in bacteria of the plasmids used in the construction ofthe clone of infective cDNA (pBAC-TcDNA^(−ΔClaI) and pBAC-ClaI^(F)), aswell as the plasmid that contains the complete cDNA (pBAC-TcDNA^(FL));was analyzed after being grown in E. coli for 160 generations. Thestability was analyzed by means of digestion with restriction enzymes ofthe purified DNAs. No deletions or insertions were detected, althoughthe presence of minor changes not detected by the analysis techniqueused cannot be ruled out in the case of the pBAC-TcDNA^(−ΔclaI) plasmidand the pBAC-B+C+D5′ plasmid. In the case of the pBAC-TcDNA plasmid,which contains the complete genome of TGEV, a certain instability wasdetected after 40 generations (at this point approximately 80% of theDNA presented the correct restriction pattern). This slight instability,however, does not represent an obstacle to the rescue of the infectivevirus, since 20 generations (4 L of culture) of bacterial growth aresufficient to generate a quantity of plasmid DNA that allows the virusto be rescued.

1.2 Rescue of an Infective TGEV from a cDNA that Codes for the completeGenome

ST cells were transfected with the pBAC-TcDNA^(FL) plasmid. At 48 hposttransfection, the supernatant of the culture was collected andpassed into ST cells six times (see FIG. 5). Starting at passage 2, at14 h postinfection, the cytopathic effect became apparent, extendinglater, at 20 h postinfection, to practically all of these cells thatformed the monolayer (see FIG. 6). On the other hand, the titer ofrescued virus increased rapidly with the passages, reaching values onthe order of 10⁸ PFU/mL as of passage 3 (see FIG. 7). The experiment wasrepeated five times, and in ail cases, infective virus with similartiters were recovered, whereas, in the case of nontransfected ST cellsor ST cells transfected with a similar plasmid, where the ClaI-ClaIfragment was found in the opposite orientation, virus was neverrecovered.

For the purpose of eliminating the possibility that the virus obtainedwas the product of contamination, the sequence at positions 6,752 and18,997 was determined by means of sequencing of cDNA fragments amplifiedby RT-PCR using the genomic RNA of the rescued virus as a template. Theanalysis of the sequence determined that the nucleotides in positions6,752 and 18,997 were those present in the cDNA. Furthermore, therescued virus presented, in the cDNA sequence of the S gene, arestriction site DraIII at position 20,990, as was expected for the Sgene of C11 (FIG. 8). The presence of these three genetic markersconfirmed that the isolated virus came from the cDNA.

In a more in-depth characterization of the virus generated, acomparative analysis was made by immunofluorescence of infected cellswith the virus recovered (TcDNA) after transfection with thepBAC-TcDNA^(FL) plasmid or cells infected with the PUR46-MAD isolate ofthe TGEV. For this, specific polyclonal and monoclonal antibodies thatrecognized both the C11 isolate and the PUR46-MAD isolate, or only thelatter, were used (see FIG. 10). The results obtained confirmed theantigenicity expected for the new TcDNA virus. The polyclonal antibodyspecific for TGEV, the expected specific monoclonal of the S protein(ID.B12 and 6A.C3), as well as the specific monoclonal of the M (3B.B3)and N (3B.D8) proteins recognized both the TcDNA and the PUR46-MAD. Thedata obtained indicated that the virus generated presented the M and Nproteins of the PUR46-MAD isolate and the S protein of the C11 isolate,as had been designed in the original cDNA.

1.3 In Vivo Infectivity and Virulence

For the purpose of analyzing the in vivo, infectivity of the TcDNAvirus, a group of five newborn pigs was inoculated with virus clonedfrom passage 6, and mortality was analyzed. The five inoculated pigsdied 3 to 4 days postinoculation, indicating that the TcDNA virus wasvirulent. In contrast, two pigs inoculated only with the diluent of thevirus and maintained in the same conditions did not suffer alterations.

1.4 Optimization of the Levels of Expression by Modification of theTranscription-Regulating Sequences

RNA synthesis in coronavirus takes place by means, of an RNA-dependentprocess, in which the mRNAs are transcribed from templates with negativepolarity. In the TGEV, a conserved consensus sequence, CUAAAC, appears,which is located just in front of the majority of the genes. Thesesequences represent signals for the transcription of the subgenomicmRNAs. In coronavirus, there are between six and eight types of mRNAswith variable sizes, depending on the type of coronavirus and of thehost. The largest corresponds to the genomic RNA, which in turn servesas mRNA for ORFs 1a and 1b. The rest of the mRNAs correspond tosubgenomic mRNAs. These RNAs are denominated mRNA 1 to 7, in decreasingsize order. On the other hand, some mRNAs that have been discoveredafter the set of originally described mRNAs have been denominated withthe name of the corresponding mRNA, a dash, and a number, e.g., mRNA2-1. The mRNAs present a coterminal structure in relation to thestructure of the genomic RNA. With the exception of the smallest mRNA,the rest are structurally polycistronic, while, in general, only the ORFlocated closest to 5′ is translated.

The efficiency in the expression of a marker gene (GUS) has been studiedusing different sequences flanking the 5′-terminal of the minimalintergenic (IG) sequence CUAAAC (FIG. 11), different sequences flankingthe 3′-terminal of the IG sequence (FIG. 12), and various insertionsites (FIG. 13). The results obtained (FIGS. 11 to 13) indicated thatoptimal expression was achieved with a TRS consisting of: (i) the −88 ntflanking the consent sequence for the N gene of TGEV; (ii) the IGsequence; and (iii) the 3′-flanking sequence of the IG sequence of the Sgene. Furthermore, in agreement with the results obtained inrelationship to the point of insertion of the heterologous gene, thegreatest levels of expression were achieved when the heterologous genewas located at the 3′-end of the genome. A TRS such as that describedallows the GUS to be expressed at levels between 2 and 8 μg per 10⁶cells.

1.5 Tissue Specificity of the System of Expression

Many pathogens enter the host through the mucous membranes. To preventthis type of infections, it is important to develop systems ofexpression that allow the induction of high levels of secretoryimmunity. This can be achieved fundamentally through the administrationof antigens in the lymph nodes associated with the respiratory andintestinal tract. To achieve this goal, and in general to direct theexpression of a gene at the tissue of interest, the molecular bases ofthe tropism of TGEV have been studied. These studies have showed thatthe tissue specificity of TGEV can be modified by the construction ofrecombinant viruses containing the S gene of coronavirus with thedesired tropism (Ballesteros et al., 1997; Sánchez et al., 1999). Thisinformation makes it possible to construct systems of expression basedon cDNA genomes of coronavirus with respiratory or intestinal tropism.

1.6 Expression of the Viral Antigen Coded by the ORF5 of PRRSV usingInfective cDNA

For the purpose of optimizing the levels of expression of heterologousgenes, constructions were made from a vector of interchangeable modulesflanked by cloning sequences that facilitate the exchange of TRSs andheterologous genes within the vector. The construction, which includedORF 5 of the PRRSV (Porcine respiratory and reproductive syndrome virus)flanked at the 5′-end by the minimal IGS consensus sequence (CUAAAC)preceded by the −88 nts flanking the gene of the viral nucleocapsid (N),and at the 3′-end by restriction site SalI (GTCGAC) and a sequenceanalogous to that of Kozak (AC)GACC, yielded an optimal expression(about 10 μg/10⁶ cells). In principle, these levels of expression of theheterologous gene are more than sufficient to induce an immune response.The heterologous gene was inserted into the position previously occupiedby genes 3a and 3b of the virus, which are dispensable.

1.7 Induction of an Immune Response in Swine to an Antigen Expressedwith the cDNA Derived Virus Vector

Using the same type of virus vector derived from the cDNA and the TRSsdescribed above, the gene encoding the green fluorescent protein (GFP)was expressed at high levels (20 μg of protein per million of cells inswine testis, ST, cells). The expression levels were stable for morethan 20 passages in cell culture. Furthermore, a set of swine wereimmunized with the live virus vector, that was administered by the oral,intranasal and intragastric routes and a strong humeral immune responsewas detected against both the virus vector and the GFP. Interestingly,no secondary effect was observed in the inoculated animals after theadministration of three doses of the virus vector.

1.8 Construction of a Safe Virus Vector that Expresses the Foreign Genewithout Leading to the Generation of an Infectious Virus.

To design vector for humans, biosafety is a priority. To achieve thisgoal, three types of safety guards are being engineered in the vector.Two of these are based on the deletion of two virus components, mappingat different positions of the virus genome, essential for thereplication of the virus. These components are being provided in transby a packaging cell line. This cell (Baby Hamster Kidney, BHK) expressesthe missing TGEV genes encoding essential structural proteins of thevirus (the envelope E and the membrane M proteins). The third safetyguard is the relocation of the packaging signal of the virus genome, insuch a way that the recovery of an infectious virus by recombination isprevented, leading to the generation of a suicide vector thatefficiently expresses the heterologous genes but that is unable topropagate even to the closest neighbor cell.

With the design of the new vector for use in humans, we are notproducing a new virus that could be propagated within the human species,since this vector can not be transmitted from cell to cell in humanbeings. The vector is based on a replication defective virus. It canonly be grown in the vaccine factory by using packaging cellscomplementing the deletions of the virus. These safety guards representnovel procedures in the engineering of coronaviruses. The recombinantvirus with a new tropism will be replication competent at least infeline cells, since these cells replicate human, porcine, canine andfeline coronaviruses.

Deposition of Microorganisms:

The bacterium derived from Escherichia coli, carrying the plasmid withthe infective clone of the invention, identified as Escherichia colipBAC-TcDNA^(FL), has been deposited with the Spanish Collection of TypeCultures (CECT), Burjassot (Valencia), on Nov. 24^(th) 1999, underregistration number CECT 5265.

BIBLIOGRAPHY

-   Ahlquist, P., R. French, M. Janda, and L. S. Loesch-Fries. (1984).    Multicomponent RNA plant virus infection derived from cloned viral    cDNA. Proc. Natl. Acad. Sci. USA. 81:7066-7070.-   Almazan, F., J. M. González, Z. Pénzes, A. Izeta, E. Calvo, J.    Plana-Durán, and L. Enjuanes. (2000). Engineering the largest RNA    virus genome as an infectious bacterial artificial chromosome. Proc.    Natl. Acad. Sci. USA. 97:5516-5521.-   Ballesteros, M. L., C. M. Sanchez, and L. Enjuanes. (1997). Two    amino acid changes at the N-terminus of transmissible    gastroenteritis coronavirus spike protein result in the loss of    enteric tropism. Virology. 227:378-388.-   Baron, M. D., and T. Barrett. (1997). Rescue of rinderpest virus    from cloned cDNA. J. Virol. 71:1265-1271.-   Boyer, J. C., A. L. and Haenni. (1994). Infectious transcripts and    cDNA clones of RNA viruses. Virology. 198:415-426.-   Chang, R. Y., M. A. Hofmann, P. B. Sethna, and D. A. Brian. (1994).    A cis-acting function for the coronavirus leader in defective    interfering RNA replication. J. Virol. 68:8223-8231.-   Collins, P. L., M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock,    and B. R. Murphy. (1995). Production of infectious human respiratory    syncytial virus from cloned dCNA confirms an essential role for the    transcription elongation factor from the 5′ proximal open reading    frame of the M2 mRNA in gene expression and provides a capability    for vaccine development. Proc. Natl. Acad. Sci. USA. 92:11563-11567.-   Davis, N. L., L. V. Willis, J. F. Smith, and R. E. Johnston. (1989).    In vitro synthesis of infectious Venezuelan equine encephalitis    virus RNA from a cDNA clone: analysis of a viable deletion mutant.    Virology. 171:189-204.-   Dubensky, J., T. W., D. A. Driver, J. M. Polo, B. A. Belli, E. M.    Latham, C. E. Ibanez, S. Chada, D. Brumm, T. A. Banks, S. J.    Mento, D. J. Jolly, and S. M. W. Chang. (1996). Sindbis virus    DNA-based expression vectors: utility for in vitro and in vivo gene    transfer. J. Virol. 70:508-519.-   Durbin, A. P., S. L. Hall, J. W. Slew, S. S. Whitehead, P. L.    Collins, and B. R. Murphy. (1997). Recovery of infectious human    parainfluenza virus type 3 from cDNA. Virology. 235:323-332.-   Enjuanes, L., S. G. Siddell, and W. J. Spaan. 1998. Coronaviruses    and Arteriviruses. Plenum Press, New York.-   Enjuanes, L., and B. A. M. Van der Zeijst. 1995. Molecular basis of    transmissible gastroenteritis coronavirus epidemiology. In The    Coronaviridae. S. G. Siddell, editor. Plenum Press, New York.    337-376.-   Frolov, I., T. A. Hoffman, B. M. Prágai, S. A. Dryga, H. V.    Huang, S. Schlesinger, and C. M. Rice. (1996). Alphavirus-based    expression vectors: Strategies and applications. Proc. Natl. Acad.    Sci. USA. 93:11371-11377.-   Garcin, D., T. Pelet, P. Calain, L. Roux, J. Curran, and D.    Kolakofsky. (1995). A highly recombinogenic system for the recovery    of infectious sendai paramyxovirus from cDNA: generation of a novel.    EMBO J. 14:6087-6094.-   Geigenmuller, U., N. H. Ginzton, and S. M. Matsui. (1997).    Construction of a genome-length cDNA clone for human astrovirus    serotype 1 and synthesis of infectious RNA transcripts. J. Virol.    71:1713-1717.-   Izeta, A., C. Smerdou, S. Alonso, Z. Penzes, A. Mendez, J.    Plana-Duran, and L. Enjuanes. (1999). Replication and packaging of    transmissible gastroenteritis coronavirus-derived synthetic    minigenomes. J. Virol. 73:1535-1545.-   Kim, U. -J., H. Shizuya, P. de Jong, B. W. Birren, and M. I. Simon.    (1992). Stable propagation of cosmid-sized human DNA inserts in an    F-factor based vector. Nucleic Acids Res. 20:1083-1085.-   Lai, C. -J., B. Zhao, H. Hori, and M. Bray. (1991). Infectious RNA    transcribed from stably cloned full-length cDNA of dengue type 4    virus. Proc. Natl. Acad. Sci. USA. 88:5139-5143.-   Lai, M. M. C., and D. Cavanagh. (1997). The molecular biology of    coronaviruses. Adv. Virus Res. 48:1-100.-   Lai, M. M. C., C. -L. Liao, Y. -J. Lin, and X. Zhang. (1994).    Coronavirus: how a large RNA viral genome is replicated and    transcribed. Infect. Agents Dis. 3:98-105.-   Liljeström, P. (1994). Alphavirus expression systems. Curr. Opin.    Biotech. 5:495-500.-   Liljeström, P., and H. Garoff. (1991). A new generation of animal    cell expression vectors based on the Semliki Forest virus replicon.    Bio/Technology. 9:1356-1361.-   Luytjes, W., M. Krystal, M. Enami, J. D. Parvin, and P. Palese.    (1989). Amplification, expression, and packaging of a foreign gene    by influenza virus. Cell. 59:1107-1113.-   Mandl, C. W., M. Ecker, H. Holzmann, C. Kunz, F. X. Heinz.    (1997).Infectious cDNA clones of tick-borne encephalitis virus    European subtype prototypic strain Neudoerfl and high virulence    strain Hypr. J. Gen. Virol. 78:1049-1057.-   Maniatis, T., E. F. Fritsh, and J. Sambrook, (1989). Molecular    cloning: a laboratory manual. Cold Spring Harbour Laboratory Press.    New York-   Méndez, A., C. Smerdou, A. Izeta, F. Gebauer, and L. Enjuanes.    (1996). molecular characterization of transmissible gastroenteritis    coronavirus defective interfering genomes: packaging and    heterogeneity. Virology. 217:495-507.-   Penzes, Z., A. Izeta, C. Smerdou, A. Mendez, M. L. Ballesteros,    and L. Enjuanes. (1999). Complete nucleotide sequence of    transmissible gastroenteritis coronavirus strain PUR46-MAD.    Submitted for publication-   Pushko, P., M. Parker, G. V. Ludwing, N. L. Davis, R. E. Johnston,    and J. F. Smith. (1997). Replication-helper systems from attenuated    Venezuelan equine encephalitis virus: expression of heterologous    genes in vitro and immunization against heterologous pathogens in    vivo. Virology. 239:389-401.-   Racaniello, V. R., and D. Baltimore. (1981). Cloned poliovirus cDNA    is infectious in mammalian cells. Science. 214:916-919.-   Radecke, F., P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, C.    Dotsch, G. Christiansen, and M. A. Billeter. (1995). Rescue of    measles viruses form cloned DNA. EMBO J. 14:5773-5784.-   Rice, C. M., A. Grakoui, R. Galler, and T. J. Chambers. (1989).    Transcription of infectious yellow fever RNA from full-length cDNA    templates produced by in vitro ligation. New Biologist. 1:285-296.-   Rice, C. N., R. Levis, J. H. Strauss, and H. V. Huang. (1987).    Production of infectious RNA transcripts from Sindbis virus cDNA    clones: Mapping of lethal mutations, rescue of a    temperature-sensitive marker, and in vitro mutagenesis to generate    defined mutants. J. Virol. 61:3809-3819.-   Rice, C. M., and J. H. Strauss. (1981). Synthesis, cleavage, and    sequence analysis of DNA complementary to the 26S messenger RNA of    Sindbis virus. J. Mol. Biol. 150:315-340.-   Ruggli, N., J. D. Tratschin, C. Mittelholzer, M. A. Hofmann. (1996).    Nucleotide sequence of classical swine fever virus strain Alfort/187    and transciption of infectious RNA from stably cloned full-length    cDNA. J. Virol. 70:3479-3487.-   Sánchez, C. M., G. Jiménez, M. D. Laviada, I. Correa, C. Suñé, M. J.    Bullido, F. Gebauer, C. Smerdou, P. Callebaut, J. M. Escribano,    and L. Enjuanes. (1990). Antigenic homology among coronaviruses    related to transmissible gastroenteritis virus. Virology.    174:410-417-   Sánchez, C. M., F. Gebauer, C. Suñé, A. Mendez, J. Dopazo, and L.    Enjuanes. (1992). Genetic evolution and tropism of transmissible    gastroenteritis coronaviruses. Virology. 190:92-105.-   Sánchez, C. M., A. Izeta, J. M. Sanchez-Morgado, S. Alonso, I.    Sola, M. Balasch, J. Plana-Durán, and L. Enjuanes. (1999). Targeted    recombination demonstrates that the spike gene of transmissible    gastroenteritis coronavirus is a determinant of its enteric tropism    and virulence. J. Virol. 73:7607-7618.-   Sawicki, S. G., and D. L. Sawicki. (1990). Coronavirus    transcription: subgenomic mouse hepatitis virus replicative    intermediates function in RNA synthesis. J. Virol. 64:1050-1056.-   Schnell, M. J., T. Mebatsion, and K. -K. Conzelmann. (1994).    Infectious rabies viruses from cloned cDNA. EMBO J. 13:4195-4203.-   Sethna, P. B., S. -L. Hung, and D. A. Brian. (1989). Coronavirus    subgenomic minus-strand RNAs and the potential for mRNA replicons.    Proc. Natl. Acad. Sci. USA. 86:5626-5630.-   Shizuya, H., B. Birren, U. -J. Kim, V. Mancino, T. Slepak, Y.    Tachiuri, and M. Simon. (1992). Cloning and stable maintenance of    300-kilobase-pair fragments of human DNA in Escherichia coli using    an F-factor-based vector. Proc. Natl. Acad. Sci. USA. 89:8794-8797.-   Siddell, S. G. 1995. The Coronaviridae. Plenum Press, New York. 418    pp.-   Smerdou, C., and P. Liljestrom. (1999). Non-viral amplification    systems for gene transfer: vectors based on alphaviruses. Curr.    Opin. Mol. Therap. 1:244-251.-   Taniguchi, M., and F. A. P. Miller. (1978). Specific suppressive    factors produced by hybridomas derived from the fusion of enriched    suppressor T cells and A T lymphoma cell line. J. Exp. Med.    148:373-382.-   van der Most, R. G., and W. J. M. Spaan. 1995. Coronavirus    replication, transcription, and RNA recombination. In The    Coronaviridae. S. G. Siddell, editor. Plenum Press, New York. 11-31.-   Wang, K., C. Boysen, H. Shizuya, M. I. Simon, and L. Hood. (1997).    Complete nucleotide sequence of two generations of a bacterial    artificial chromosome cloning vector. BioTechniques. 23:992-994.-   Woo, S. -S., J. Jiang, B. S. Gill, A. H. Paterson, and R. A. Wing.    (1994). Construction and characterization of a bacterial artificial    chromosome library of Sorghum bicolor. Nucleic Acids Res.    22:4922-4931.-   Zhang, X., C. L. Liao, and N. M. C. Lai. (1994). Coronavirus leader    RNA regulates and initiates subgenomic mRNA transcription both in    trans and in cis. J. Virol. 68:4738-4746.

1. A bacterial artificial chromosome construct comprising a nucleic acidsequence that directs formation of a recombinant coronavirus uponintroduction into a cell.
 2. The artificial chromosome construct ofclaim 1, wherein said artificial chromosome or said nucleic acidsequence further comprises a heterologous nucleic acid sequence.
 3. Theartificial chromosome construct of claim 2, wherein said heterologousnucleic acid sequence encodes a therapeutic gene product.
 4. Theartificial chromosome construct of claim 3, wherein said therapeuticgene product is a protein, a peptide, or an epitope.
 5. The artificialchromosome construct of claim 3, wherein said therapeutic gene productis selected from the group consisting of a ribozyme, an antigen from aninfectious agent, a molecule interfering with the replication of aninfectious agent, an antibody, an immune modulator, a cytokine, animmunoenhancer, an anti-inflammatory compound, an enzyme, a substancethat potentiates subpopulations of helper T-cells, cellular necrosisfactor, and substances that provoke cellular immunity.